Infection and propagation of prions requires not only expression of the prion protein (PrP) gene encoded by the mammalian host, but also the cooperation of other host genes (i.e. non-PrP genes for prion propagation/pathogenicity; NPGPPs). There is evidence that the protein products of NPGPPs assist in the folding of conformationally altered prion protein (PrP so) and they additionally may assist in the uptake, internalization, and transport of PrPSc in infected cells. The proposed research will employ a novel mutational approach (random homozygous knockout; RHKO), which previously has been used successfully for the discovery of host genes needed for viral infection, to identify NPGPPs required for the propagation of prions. Its objective is to construct RHKO libraries in cell lines infectable by prions in culture, isolate cell clones resistant to prion infection and/or pathogenicity, identify non-prion cellular genes (i.e., NPGPPs) whose functional inactivation by RHKO has resulted in resistance, and use NPGPP-mediated alterations in gene expression fingerprints to identify cellular functions and pathways affected by NPGPPs. The research, which will use a combination of genetic and biochemical experimental tools, will provide important new information about prion-induced age-dependent neurodegeneration. Additionally, it is expected to establish a foundation for subsequent studies of mechanisms involved in prion pathogenicity, yield validated targets for potential therapeutic agents that may interfere with prion propagation, and provide a possible genetic framework for approaches aimed at breeding livestock unable to propagate prions.